SOLUBILIZED TETRODOTOXIN BINDING-COMPONENT FROM THE ELECTROPLAX OF ELECTROPHORUS-ELECTRICUS - STABILITY AS A FUNCTION OF MIXED LIPID-DETERGENT MICELLE COMPOSITION

In an attempt to provide a description of the molecular components responsible for the propagated action potential, we have investigated the solubilization of the voltage-sensitive sodium channel from the electroplax membranes of Electrophorus electricus. This channel is similar to those from nerve and muscle membranes and may be quantified by the binding of radiolabeled tetrodotoxin. When electroplax membranes were treated with Lubrol-PX, a tetrodotoxin binding protein was released in soluble form. The solubilized protein was extremely unstable to storage, elevated temperature, mild fractionation procedures, and elevated detergent concentrations. Loss of binding activity, measured at 18 °C, followed essentially a first-order exponential dependence on time, and the apparent first-order decay constant (kr) was measured as an index of instability. In the initial extract kT was a quantitatively reproducible function of the ratio of endogenous lipid phosphate to detergent. This ratio could be altered by (1) increasing the amount of detergent while holding the final concentration of extract constant, (2) dilution of extract into buffer containing a constant final concentration of detergent, or (3) addition of high concentrations of detergent but in combination with variable amounts of a lipid preparation from electroplax membranes. In all cases kT depended only on the final ratio of lipid phosphate to detergent. When this molar ratio fell below about 0.07, the protein became enormously unstable. Experiments with pure lipids indicated that not all species were equally effective for stabilization. It has been concluded that the pure detergent micelle does not reproduce the stabilizing environment encountered in the native membrane but that this can be achieved by including low molar ratios of certain pure lipids such as phosphatidylcholine. The extreme instability observed at low lipid-detergent ratios indicates that the protein may not be easily purified or characterized unless mixed lipid-detergent micelles are employed. © 1979, American Chemical Society. All rights reserved.